Lignocellulosic biomass represents one of the most abundant renewable resources on earth, and certainly one of the least expensive. The substrates considered are very varied since they concern both lignous substrates (broadleaved trees and coniferous trees), agricultural sub-products (straw) or sub-products from industries generating lignocellulosic waste (food-processing industries, paper industries).
Lignocellulosic biomass consists of three main polymers: cellulose (35 to 50%), hemicellulose (20 to 30%), which is a polysaccharide essentially consisting of pentoses and hexoses, and lignin (15 to 25%), which is a polymer of complex structure and high molecular weight, consisting of aromatic alcohols linked by ether bonds.
These various molecules are responsible for the intrinsic properties of the plant cell walls and they organize into a complex entanglement.
The cellulose and possibly the hemicelluloses are the targets of enzymatic hydrolysis, but they are not directly accessible to enzymes. These substrates therefore have to undergo a pretreatment prior to the enzymatic hydrolysis stage. The pretreatment aims to modify the physical and physico-chemical properties of the lignocellulosic material in order to improve the accessibility of the cellulose stuck in the lignin and hemicellulose matrix. It can also release the sugars contained in the hemicelluloses as monomers, essentially pentoses, such as xylose and arabinose, and hexoses, such as galactose, mannose and glucose.
Ideally, the pretreatment must be fast and efficient, with high substrate concentrations, and material losses should be minimal. There are many technologies available: acidic boiling, alkaline boiling, steam explosion (Pourquié J. and Vandecasteele J. P. (1993) Conversion de la biomasse lignocellulosique par hydrolyse enzymatique et fermentation. Biotechnologie, 4th ed., René Scriban, coordinateur Lavoisier TEC & DOC, Paris, 677-700), Organosolv processes, or twin-screw technologies combining thermal, mechanical and chemical actions (Ogier J. C. et al. (1999) Production d'éthanol à partir de biomasse lignocellulosique, Oil & Gas Science & Technology 54:67-94). The pretreatment efficiency is measured by the hydrolysis susceptibility of the cellulosic residue and by the hemicellulose recovery rate. From an economic point of view, the pretreatment preferably leads to total hydrolysis of the hemicelluloses, so as to recover the pentoses and possibly to upgrade them separately from the cellulosic fraction. Acidic pretreatments under mild conditions and steam explosion are well suited techniques. They allow significant recovery of the sugars obtained from the hemicelluloses and good accessibility of the cellulose to hydrolysis.
The cellulosic residue obtained is hydrolyzed via the enzymatic process using cellulolytic and/or hemicellulolytic enzymes. Microorganisms such as fungi belonging to the Trichoderma, Aspergillus, Penicillium or Schizophyllum genera, or anaerobic bacteria belonging for example to the Clostridium genus, produce these enzymes containing notably cellulases and hemicellulases, suited for total hydrolysis of the cellulose and of the hemicelluloses.
Enzymatic hydrolysis is carried out under mild conditions (temperature of the order of 45-50° C. and pH value 4.8) and it is efficient. On the other hand, as regards the process, the cost of enzymes is still very high. Considerable work has therefore been conducted in order to reduce this cost: i) first, increase in the production of enzymes by selecting hyperproductive strains and by improving fermentation methods, then ii) decrease in the amount of enzymes in hydrolysis by optimizing the pretreatment stage or by improving the specific activity of these enzymes. During the last decade, the main work consisted in trying to understand the mechanisms of action of the cellulases and of expression of the enzymes so as to cause excretion of the enzymatic complex that is best suited for hydrolysis of the lignocellulosic substrates by modifying the strains with molecular biology tools.
Filamentous fungi, as cellulolytic organisms, are of great interest to industrialists because they have the capacity to produce extracellular enzymes in very large amounts. The most commonly used microorganism for cellulase production is the Trichoderma reesei fungus. Wild strains have the ability to produce, in the presence of an inductive substrate, cellulose for example, a secretome (all the proteins secreted) suited for cellulose hydrolysis. The enzymes of the enzymatic complex comprise three major types of activities: endoglucanases, exoglucanases and β-glucosidases. Other proteins having properties that are essential for the hydrolysis of lignocellulosic materials are also produced by Trichoderma reesei, xylanases for example. The presence of an inductive substrate is essential to the expression of cellulolytic and/or hemicellulolytic enzymes. The nature of the carbon-containing substrate has a strong influence on the composition of the enzymatic complex. It is the case of xylose which, associated with an inductive carbon-containing substrate such as cellulose or lactose, allows the activity referred to as xylanase activity to be significantly improved.
Conventional genetic techniques using mutagenesis have allowed cellulase-hyperproductive Trichoderma reesei strains such as MCG77 (Gallo—U.S. Pat. No. 4,275,167), MCG 80 (Allen, A. L. and Andreotti, R. E., Biotechnol-Bioengi 1982, 12, 451-459 1982), RUT C30 (Montenecourt, B. S. and Eveleigh, D. E., Appl. Environ. Microbiol. 1977, 34, 777-782) and CL847 (Durand et al., 1984, Proc. Colloque SFM “Génétique des microorganismes industriels”. Paris. H. HESLOT Ed, pp 39-50) to be selected. The improvements have allowed to obtain hyperproductive strains that are less sensitive to catabolic repression on monomer sugars notably, glucose for example, in relation to wild strains.
The fact that genetic techniques intended to express heterologous genes within these fungic strains are now widely practised has also opened up the way for the use of such microorganisms as hosts for industrial production. New techniques of studying enzymatic profiles have made it possible to create very efficient host fungic strains for the production of recombining enzymes on the industrial scale [Nevalainen H. and Teo V. J. S. (2003) Enzyme production in industrial fungi-molecular genetic strategies for integrated strain improvement. In Applied Mycology and Biotechnology (Vol. 3) Fungal Genomics (Arora D. K. and Kchachatourians G. G. eds.), pp. 241-259, Elsevier Science]. One example of this type of modification is the production of cellulases from a T. reesei strain [Harkki A. et al. (1991) Genetic engineering of Trichoderma to produce strains with novel cellulase profiles. Enzyme Microb. Technol. 13, 227-233; Karhunen T. et al. (1993) High-frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. Mol. Gen. Genet. 241, 515-522].
The sugars obtained by lignocellulosic biomass hydrolysis are pentoses (mainly xylose and arabinose), disaccharides (cellobiose) and glucose. The latter is readily converted to ethanol by the yeast S. cerevisiae used by all the alcoholic fermentation industries. Currently, no other microorganism reaches its performances on glucose under non-sterile conditions, i.e. a yield of the order of 0.47 g ethanol per gram of glucose, a productivity greater than or equal to 5 g/l×h, and final ethanol concentrations close to 10% by volume. S. cerevisiae affords many additional advantages resulting from many years of selection: resistance to ethanol, easy industrial implementation, etc. On the other hand, pentoses are rarely fermented by microorganisms, and when they are, the performances are poor. During the past years, considerable work has been done on the search for and/or the improvement of strains providing active fermentation of pentoses to ethanol. Four types of microorganisms have been studied: the yeasts fermenting pentoses naturally, recombined S. cerevisiae strains, thermophilic or mesophilic bacteria using pentoses.
Alcoholic fermentation under non-sterile conditions involves a high risk of contamination, of the fermenter by opportunistic microorganisms. The contamination sources can be of living or non-living nature. However, we shall only deal here with living contamination sources. These sources mainly include yeasts and bacteria. These microorganisms use the nutrients that are present, including the carbon source, and they are responsible for the formation of unwanted co-products such as lactic acid, acetic acid or even acetone and butanol. This kind of microorganisms is found wherever the conditions allow their growth, i.e. In the presence of a minimum amount of nutrients. It can be mentioned here that their nutritional requirements are: a source of carbon (generally sugars), a source of amino-acids (constituents of proteins), some vitamins and trace elements.
Furthermore, within the energetic raw materials considered, wheat straw for example, it is likely that microorganisms capable of contaminating the process can be found.
Thus, in the current and non-sterile method of producing second-generation ethanol, two stages are sensitive to a possible microbiological contamination: the enzymatic hydrolysis stage and the fermentation stage. The solutions currently known for fighting lactic contamination consist in lowering the pH value down to a value promoting the development of yeasts to the detriment of lactic bacteria. Yeasts are however less active at such acidic pH values. Another option consists in introducing bacterial contamination inhibitors, such as fluorine, antibiotics or sulfites, during the alcoholic fermentation stage. It is indeed during this stage of the method that the contaminant concentration is the highest. Using conventional antibacterial agents is relatively expensive and requires rather frequent fermentation process restarting procedures.
Limiting contamination risks potentially allows to save time and money for industrial processes of such scale and no solution should be neglected to overcome this problem.
Bacterial contamination is in fact a major problem in the production of ethanol by fermentation. Bacteria are naturally present within the production tool and they use the nutrients present in the medium, thus consequently competing with the yeasts used in the process. The growth and the viability of the yeast cells are therefore greatly affected by the presence of these bacteria and the final alcohol yield is also reduced thereby.
In general, lactic bacteria ferment sugars present in the fermentation musts and their growth is promoted by anaerobic conditions. They generally develop at a pH value of 5.5 but they can survive at a pH value as low as 3.0. These opportunistic bacteria can develop over a wide temperature range and they are tolerant to high alcohol concentrations in the medium. The presence of bacteria in second-generation ethanol production processes should be proscribed. Any improvement in the process leading to maximum limitation of this contamination has to be taken into consideration.